Angiotensin converting enzyme 2 (ACE2) is a mediator of renal and cardiovascular function reflected by its capability to eliminate the potent vasoconstrictor angiotensin II (Ang II) by generating the vasodilator angiotensin (1-7) (Ang (1-7)). ACE2 signaling is critical in promoting cardiovascular and renal health especially under pathological conditions. Deletion of ACE2 produces a mouse phenotype which is essentially normal with respect to basal renal and cardiac function, suggesting an activation of compensatory mechanisms. Indeed, studies have shown that Ang (1-7) is present and actually synthesized in the kidney of ACE2 knock out (ACE2KO) mice. The proposed experiments will work towards understanding the role of ACE2 and Ang (1-7) in renal and cardiovascular function under normal and pathological conditions. The question is what are the feedback mechanisms compensating for ACE2 deficiency in ACE2KO mice thus causing a normal phenotype. Compensatory mechanisms will be characterized by utilizing a newly established, sensitive and highly specific mass spectrometric imaging method, MALDI imaging. This powerful technique will be applied to identify Ang (1-7) generation in the renal cortex. Strategic use of peptide substrates and specific inhibitors will assign enzymatic activity to one of the two other known peptidases capable of forming Ang (1-7) from Ang II, prolyl oligopeptidase and prolyl carboxypeptidase. Real time PCR, Western Blot and immunohistochemical techniques will correlate enzyme activity with gene and protein levels. The response of these compensatory mechanisms, in particular activities of prolyl oligopeptidase and prolyl carboxypeptidase as well as Ang II/Ang (1-7) peptide levels, will be further characterized after chronic infusion of Ang II, a pathological insult known to cause hypertension and renal damage. The resulting changes in cardiovascular and renal function will be monitored by radiotelemetric blood pressure measurements, urinary volume and albumin/creatinine excretion, histological examination of kidney tissue and Ang II/Ang (1-7) peptide level analysis in urine, blood and kidney. Identification of molecular mechanisms compensating ACE2 deficiency may contribute to a better understanding of the causes of renal and cardiovascular pathologies and may aid towards the development of pharmacological strategies against these diseases. PUBLIC HEALTH RELEVANCE: There is much need for new strategies to prevent kidney and cardiovascular disease, leading causes of death in the U.S.A. This research program uses newly developed, state-of-the-art tissue imaging techniques to examine the mechanisms by which local peptide hormones regulate kidney/cardiovascular function. The goal is to uncover new therapeutic approaches which may target the diseases.